Nr4a1 and Nr4a3 Reporter Mice Are Differentially Sensitive to T Cell Receptor Signal Strength and Duration

Abstract

Summary Nr4a receptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3 -Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1 – Nr4a3 are Src family kinase dependent. Moreover, Nr4a2 and Nr4a3 are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2 and Nr4a3, but redundancy for Nr4a1 . Nr4a1- GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3- Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3- Tocky, Nr4a1- GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo .

Application of dual Nr4a1-GFP Nr4a3-Tocky reporter mice to study T cell receptor signaling by flow cytometry

“…The protocol is optimized for use of “homozygous” Nr4a3 -Tocky BAC and “heterozygous” Nr4a1 -GFP mice. CRITICAL: Use of heterozygous Nr4a3 -Tocky in combination with Nr4a1 -GFP leads to reduced expression of Nr4a3-Timer ( Jennings et al, 2020 ). Therefore, it is strongly recommended that Nr4a3 -Tocky homozygous mice are used when these are mated to Nr4a1 -GFP mice.…”

“… Use unstimulated Nr4a3-Tocky T cells and gate on Nr4a3-Blue − Nr4a3-Red + to generate a single Red color control. Note: Figure 2 shows that Blue, Red and GFP colors show no overlap (see also Table 1 )

Figure 2 GFP, Timer-Blue, and Timer-Red show no spectral overlap (A) Thymocytes from OTI Nr4a3 -Tocky Nr4a1 -GFP mice (which do not express Nr4a3-Timer in the thymus; Jennings et al, 2020 ) were analyzed for expression of Nr4a3-Blue versus Nr4a1-GFP and Nr4a3-Red versus Nr4a1-GFP channels. (B) Splenocytes from Nr4a3 -Tocky (pure) line were analyzed for Nr4a1-GFP versus Nr4a3-Red (left) or stimulated for 3 h with anti-CD3 and then Nr4a3-Blue + Nr4a3-Red − cells analyzed for Nr4a1-GFP versus Nr4a3-Blue.

…”

“… (A) Thymocytes from OTI Nr4a3 -Tocky Nr4a1 -GFP mice (which do not express Nr4a3-Timer in the thymus; Jennings et al, 2020 ) were analyzed for expression of Nr4a3-Blue versus Nr4a1-GFP and Nr4a3-Red versus Nr4a1-GFP channels. …”

“…It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al, 2020 .…”

“…For complete details on the use and execution of this protocol, please refer to Jennings et al, 2020 .…”